Transcription activation is typically carried out by soluble proteins engaging basal elements of the transcription apparatus - including promoter DNA and RNA polymerase - in the bacterial cytoplasm. In the Gram negative pathogen Vibrio cholerae, virulence gene expression is under control of an unusual set of membrane proteins. We hypothesize that a membrane complex including two activators, ToxR and TcpP, binds to the toxT promoter, recruits RNA polymerase, and activates toxT gene expression leading to activation of ToxT-controlled virulence genes. The mechanism by which membrane proteins can access DNA in the cell and recruit RNA polymerase has not been uncovered with standard genetic and biochemical approaches. Single-molecule imaging methods with nanometer-scale resolution now make it possible to investigate this mechanism in living cells, and these techniques will be applied to the ToxR/TcpP system to test specific hypotheses. This exploratory proposal has the following two specific aims: 1. Construct Vibrio cholerae strains expressing photo-activatable fluorescent fusion proteins of ToxR and TcpP, and mark toxT promoter DNA in the V. cholerae genome using the lacO operator site for binding of a LacI-EYFP fusion protein. 2. Carry out single-molecule super-resolution imaging in live cells to test specific hypotheses about the mechanism and dynamics by which membrane activators bind to toxT promoter DNA for activation of virulence gene expression.